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Mannitol as an osmotic antagonist to dimethyl sulfoxide
CRYOBIOLOGY Vol. 1, No. 4, 1965
MANNITOL
AS AN OSMOTIC ANTAGONIST DIMETHYL SULFOXIDE* HENDRICK
Department
TO
B. BARNERt
of Surgery, University of Rochester School of Medicine Rochester,
On replant&ion of a frozen-thawed canine organ protected with dimethyl sulfoxide (DMSO) or glycerol, there is rapid swelling of the organ which is in part due t,o the osmotic activity of the additive. Five minutes after revascularieation of the kidney there is extreme firmness to touch, the cortex bulges through the cut capsule, renal vein flow is drastically reduced or ceases, and massive edema of the ureter ensues. In 30 to 40 min the ureteral edema begins to resolve, as does small bowel edema in this situation. However, there is no apparent improvement of the renal edema during several hours of observation. Capsulotomy, capsulectomy, and intrarenal artery injection of papaverine do not alter the sequence of events. Recognition of the magnitude of fluid shifts due to the osmotic potency of glycerol has been previously reported.2 Dimethyl sulfoxide, apparently because of its smaller molecular size and properties as a solvent, crosses cell membranes more rapidly than glycerol and, therefore, is osmotically less potent. When a kidney is perfused with a balanced salt solution containing colloid and 15% (v/v) DMSO, the initial weight loss due t,o dehydration is only one-half that occurring when a similar solution with 15% (v/v) glycerol is used (Cady, Rivers, Barner, and Watkins, unpublished observations). Figure I illustrates the dehydrating effect when isolated kidneys are perfused with these solutions. When the same kidneys are perfused with a balanced salt solution containing colloid, t.here is an immediate and rapid weight gain. In kidneys initially perfused with the DMSO-containing solution, the subsequent weight’ gain is 10% of control weight, while kidneys initially perfused with the glycerol-containing solution gain 30% R.eceived December 14, 1964. * Supported by Grant I-GS-103 from the U. S. Public Health Service. t Postdoctoral Research Fellow, National Institutes of Health.
and Dentistry,
New York
of control weight. Because cell membranes are more permeable to DMSO than glycerol, there is less dehydration when a kidney is perfused with a solution containing DMSO, and there is less weight gain when an organ containing DMSO is reimplanted and perfused with blood. When these two substances were compared wit.h the frozen-thawed, replanted canine kidney as the experimental model, there were again remarkable differences. Kidneys protected with glycerol showed only survival of renal vascular, neural, and capsular elements with rare survival of tubular cells, and no survival of the ureter (Cady et al., unpublished observations). When DMSO was used, t.here was survival of a few tubular segments and glomeruli and 45% of ureters with peristalsis noted in several of the ureters.1 Despite this improvement in cellular survival with the use of DMSO, the immediate, gross observations suggested that there was still a strong osmotic element that was acting adversely to produce severe renal swelling. The use of an intravascular osmotic agent to counteract the intrarenal DMSO seemed logical. Mannitol was selected because of its lack of toxicity and relatively slow leakage from the vascular compartment. METHOD
This protocol has been utilized in over 100 renal transplants and is well standardized with mannitol as the variable in this experiment. Five mongrel dogs weighing 8 to 10 kg underwent left nephrectomy, including a 6-cm segment of ureter. The renal artery was immediately cannulated and the kidney was perfused with standard dextran containing 15% (v/v) DMSO at 20°C for 15 min. After being placed in a sterile rubber glove, the kidney was deposited in a freezer at -10” or -50°C. After storage for 20 to 144 hr, the kidney was allowed to thaw during aubotransplantation to the right iliac fossa. Table 1 depicts the parameters of frozen storage. Im-
292
MANNITOL
AS OSMOTIC
40
I. ‘-15% Dimethyl Sulfoxlde Control in BSS 8 5% Humon ’ Balanced Electrolyte Weight Albumin ----,I -and 5% -+ t -.-15% Glycerol in BSSl 30 c & 5% Humon Albumin 25 A %
lot-----
”
”
60 Time (Minutes)
’
’
”
’
120
FIG. 1. Percentage change of control weight in isolated canine kidneys perfused with the solutions indicated. Modified from unpublished results of B. Cady, R. J. Rivers, Jr., H. B. Barner, and E. Watkins, Jr.
TABLE No.
1
Storage Temperature
Storage Time
“C
hr
2 3
-50 -50 -50
5
-10 -10
72 48 144 20 20
mediately before vascularisation of the kidney, 12.5 g of mannitol as a 25% solution were given intravenously. Qbservations were then made on the response of renal vein flow, renal swelling, cortical color, and ureteral edema over a I- to 2-hr period. The ureter was implanted in the bladder. The animals were explored 13 to 34 days later so that gross and histological observations could be made. RESULTS
Renal swel1in.g seemed less severe than in the previous studies, although this was a very subjective observation. Objectively there were multiple punctate or larger pink areas on the renal surface in contrast to the usual, diffuse blue-black color. Renal vein flow was depressed but persisted.
ANTAGONIST
TO DMSO
293
Ureteral edema was definitely decreased, in contrast to prior observations. At exploration there was capsular thickening and firmness of the kidney. The renal artery and vein were patent. Peristalsis of four of the ureters was noted, and the fifth was a hydroureter, resulting from distal obstruction. On cut section, the renal cortex was gray to yellow, very firm with accentuation of the tubular striations, and reduced in thickness by one-third. The medulla was nearly normal in color and consistency. Histologically the ureters were normal. Many glomeruli and tubules were preserved in each necrosis involved kidney, while coagulation others. Periglomerular fibrosis, fusion of the glomerular tufts, apparent thickening of the glomerular capillary basement membrane, and variable hyalinization of many of the glomeruli were noted. Tubular atrophy was common, while some tubules were dilated and contained homogeneous pink-staining material. There was no reason to believe that renal function existed unless the dilated tubules and hydroureter could be construed as evidence for function of a few nephrons. Cellular infiltrate was lacking except for a few scattered chronic inflammatory cells. Pathological changes were relatively constant and did not vary with the temperature or duration of freezing. DISCUSSION
Efforts to achieve organ preservation in the frozen state have been confined primarily to the canine kidney. Initially, glycerol was utilized as the protective additive, but DMSO has been found to be superior. Despite failure t’o achieve organ preservation, it has been shown that cells of all types associated with the kidney will survive the freeze-thaw insult. Therefore, it has seemed reasonable to pursue these efforts. At present the problem appears to be one of a vascular block that, develops minutes after revascularization of the transplant. Renal edema due to the osmotic effects of DMSO and increased vascular permeability after freezing and thawing in the face of a limiting capsule could lead to secondary small vessel closure as tissue pressure exceeded intravascular pressure. Small vessel obstruction due to endothelial swelling and vasoparalysis related to intramural edema of muscular vessels could contribute to the vascular congestion.
294
H. B. BARNER
To counteract the osmotic activity of the intraparenchymal DMSO, we have used a constant dose of mannitol in dogs of mildly varying weight. Immediate observations have shown the benefit of this program, which has been confirmed by subsequent histological observations. At the present dosage of mannitol, the osmotic strength of the intravascular compartment is much less than that of the extravascular renal compartment. To achieve isosmotic strength would require direct renal artery infusion of an osmotically active substance to avoid the severe fluid shifts and vascular compartment expansion accompanying this degree of hyperosmolarity in the total blood volume. It has seemed best to avoid post-thawing renal perfusion with solutions of graded osmolarity in an attempt to remove the additive. We have favored replantation of the organ as rapidly as possible with manipulation of the vascular compartment.
SUMMARY Canine kidneys frozen with dimethyl sulfoxide at -10” or -50°C for 24 to 144 hr and then autotransplanted have shown uniform ureteral survival and preservation of numerous tubules and glomeruli without apparent function. This success has been achieved with the intravascular administration of mannitol on vascularization of the transplant and is compared with a previously reported series in which there was 45% ureteral survival and rare survival of tubular cells when mannitol was not used. REFERENCES 1. Barner, H. B., Rivers, R. J., Jr., Cady, B., and Watkins, E., Jr. Survival of the canine ureter after freezing. Surgery, 53: 344-347, 1963. 2. Rivers, R. J., Jr., Cady, B., Barner, H. B., Haynes, L. L., and Watkins, E., Jr. Osmotic cell damage during glycerol perfusion of the isolated canine kidney. Surg. Forum, 12: 134-136, 1961.
MANNITOL
AS AN OSMOTIC ANTAGONIST DIMETHYL SULFOXIDE* HENDRICK
Department
TO
B. BARNERt
of Surgery, University of Rochester School of Medicine Rochester,
On replant&ion of a frozen-thawed canine organ protected with dimethyl sulfoxide (DMSO) or glycerol, there is rapid swelling of the organ which is in part due t,o the osmotic activity of the additive. Five minutes after revascularieation of the kidney there is extreme firmness to touch, the cortex bulges through the cut capsule, renal vein flow is drastically reduced or ceases, and massive edema of the ureter ensues. In 30 to 40 min the ureteral edema begins to resolve, as does small bowel edema in this situation. However, there is no apparent improvement of the renal edema during several hours of observation. Capsulotomy, capsulectomy, and intrarenal artery injection of papaverine do not alter the sequence of events. Recognition of the magnitude of fluid shifts due to the osmotic potency of glycerol has been previously reported.2 Dimethyl sulfoxide, apparently because of its smaller molecular size and properties as a solvent, crosses cell membranes more rapidly than glycerol and, therefore, is osmotically less potent. When a kidney is perfused with a balanced salt solution containing colloid and 15% (v/v) DMSO, the initial weight loss due t,o dehydration is only one-half that occurring when a similar solution with 15% (v/v) glycerol is used (Cady, Rivers, Barner, and Watkins, unpublished observations). Figure I illustrates the dehydrating effect when isolated kidneys are perfused with these solutions. When the same kidneys are perfused with a balanced salt solution containing colloid, t.here is an immediate and rapid weight gain. In kidneys initially perfused with the DMSO-containing solution, the subsequent weight’ gain is 10% of control weight, while kidneys initially perfused with the glycerol-containing solution gain 30% R.eceived December 14, 1964. * Supported by Grant I-GS-103 from the U. S. Public Health Service. t Postdoctoral Research Fellow, National Institutes of Health.
and Dentistry,
New York
of control weight. Because cell membranes are more permeable to DMSO than glycerol, there is less dehydration when a kidney is perfused with a solution containing DMSO, and there is less weight gain when an organ containing DMSO is reimplanted and perfused with blood. When these two substances were compared wit.h the frozen-thawed, replanted canine kidney as the experimental model, there were again remarkable differences. Kidneys protected with glycerol showed only survival of renal vascular, neural, and capsular elements with rare survival of tubular cells, and no survival of the ureter (Cady et al., unpublished observations). When DMSO was used, t.here was survival of a few tubular segments and glomeruli and 45% of ureters with peristalsis noted in several of the ureters.1 Despite this improvement in cellular survival with the use of DMSO, the immediate, gross observations suggested that there was still a strong osmotic element that was acting adversely to produce severe renal swelling. The use of an intravascular osmotic agent to counteract the intrarenal DMSO seemed logical. Mannitol was selected because of its lack of toxicity and relatively slow leakage from the vascular compartment. METHOD
This protocol has been utilized in over 100 renal transplants and is well standardized with mannitol as the variable in this experiment. Five mongrel dogs weighing 8 to 10 kg underwent left nephrectomy, including a 6-cm segment of ureter. The renal artery was immediately cannulated and the kidney was perfused with standard dextran containing 15% (v/v) DMSO at 20°C for 15 min. After being placed in a sterile rubber glove, the kidney was deposited in a freezer at -10” or -50°C. After storage for 20 to 144 hr, the kidney was allowed to thaw during aubotransplantation to the right iliac fossa. Table 1 depicts the parameters of frozen storage. Im-
292
MANNITOL
AS OSMOTIC
40
I. ‘-15% Dimethyl Sulfoxlde Control in BSS 8 5% Humon ’ Balanced Electrolyte Weight Albumin ----,I -and 5% -+ t -.-15% Glycerol in BSSl 30 c & 5% Humon Albumin 25 A %
lot-----
”
”
60 Time (Minutes)
’
’
”
’
120
FIG. 1. Percentage change of control weight in isolated canine kidneys perfused with the solutions indicated. Modified from unpublished results of B. Cady, R. J. Rivers, Jr., H. B. Barner, and E. Watkins, Jr.
TABLE No.
1
Storage Temperature
Storage Time
“C
hr
2 3
-50 -50 -50
5
-10 -10
72 48 144 20 20
mediately before vascularisation of the kidney, 12.5 g of mannitol as a 25% solution were given intravenously. Qbservations were then made on the response of renal vein flow, renal swelling, cortical color, and ureteral edema over a I- to 2-hr period. The ureter was implanted in the bladder. The animals were explored 13 to 34 days later so that gross and histological observations could be made. RESULTS
Renal swel1in.g seemed less severe than in the previous studies, although this was a very subjective observation. Objectively there were multiple punctate or larger pink areas on the renal surface in contrast to the usual, diffuse blue-black color. Renal vein flow was depressed but persisted.
ANTAGONIST
TO DMSO
293
Ureteral edema was definitely decreased, in contrast to prior observations. At exploration there was capsular thickening and firmness of the kidney. The renal artery and vein were patent. Peristalsis of four of the ureters was noted, and the fifth was a hydroureter, resulting from distal obstruction. On cut section, the renal cortex was gray to yellow, very firm with accentuation of the tubular striations, and reduced in thickness by one-third. The medulla was nearly normal in color and consistency. Histologically the ureters were normal. Many glomeruli and tubules were preserved in each necrosis involved kidney, while coagulation others. Periglomerular fibrosis, fusion of the glomerular tufts, apparent thickening of the glomerular capillary basement membrane, and variable hyalinization of many of the glomeruli were noted. Tubular atrophy was common, while some tubules were dilated and contained homogeneous pink-staining material. There was no reason to believe that renal function existed unless the dilated tubules and hydroureter could be construed as evidence for function of a few nephrons. Cellular infiltrate was lacking except for a few scattered chronic inflammatory cells. Pathological changes were relatively constant and did not vary with the temperature or duration of freezing. DISCUSSION
Efforts to achieve organ preservation in the frozen state have been confined primarily to the canine kidney. Initially, glycerol was utilized as the protective additive, but DMSO has been found to be superior. Despite failure t’o achieve organ preservation, it has been shown that cells of all types associated with the kidney will survive the freeze-thaw insult. Therefore, it has seemed reasonable to pursue these efforts. At present the problem appears to be one of a vascular block that, develops minutes after revascularization of the transplant. Renal edema due to the osmotic effects of DMSO and increased vascular permeability after freezing and thawing in the face of a limiting capsule could lead to secondary small vessel closure as tissue pressure exceeded intravascular pressure. Small vessel obstruction due to endothelial swelling and vasoparalysis related to intramural edema of muscular vessels could contribute to the vascular congestion.
294
H. B. BARNER
To counteract the osmotic activity of the intraparenchymal DMSO, we have used a constant dose of mannitol in dogs of mildly varying weight. Immediate observations have shown the benefit of this program, which has been confirmed by subsequent histological observations. At the present dosage of mannitol, the osmotic strength of the intravascular compartment is much less than that of the extravascular renal compartment. To achieve isosmotic strength would require direct renal artery infusion of an osmotically active substance to avoid the severe fluid shifts and vascular compartment expansion accompanying this degree of hyperosmolarity in the total blood volume. It has seemed best to avoid post-thawing renal perfusion with solutions of graded osmolarity in an attempt to remove the additive. We have favored replantation of the organ as rapidly as possible with manipulation of the vascular compartment.
SUMMARY Canine kidneys frozen with dimethyl sulfoxide at -10” or -50°C for 24 to 144 hr and then autotransplanted have shown uniform ureteral survival and preservation of numerous tubules and glomeruli without apparent function. This success has been achieved with the intravascular administration of mannitol on vascularization of the transplant and is compared with a previously reported series in which there was 45% ureteral survival and rare survival of tubular cells when mannitol was not used. REFERENCES 1. Barner, H. B., Rivers, R. J., Jr., Cady, B., and Watkins, E., Jr. Survival of the canine ureter after freezing. Surgery, 53: 344-347, 1963. 2. Rivers, R. J., Jr., Cady, B., Barner, H. B., Haynes, L. L., and Watkins, E., Jr. Osmotic cell damage during glycerol perfusion of the isolated canine kidney. Surg. Forum, 12: 134-136, 1961.